1. Field of the Invention
The present invention relates to onco-fetal antigens, monoclonal antibodies which are immunoreactive therewith, methods of preparation of these antibodies, and uses therefor, such as, for example, in cancer diagnosis and tumor typing.
2. Description of the Background Art
Cancer is one of humanity's most frightening and frustrating disease states. It is clear that the increased longevity of civilized man has greatly increased the probability that an individual will be afflicted with cancer. Cancer biologists are increasingly frustrated because most of the therapies available for the control of cancer, once it is clinically manifested, are at best palliative for the major killer forms thereof. Survival indexes have generally not improved among the major cancers that afflict man in spite of the billions of dollars in research and the millions of man-hours expended to develop new methods for cancer control. It is natural then that oncologists and tumor biologists have been desperately searching for better tools to detect cancer and to improve therapy based on a better understanding of cancer biology.
A small but significant number of spontaneous remissions that occur in otherwise untreatable tumors has encouraged biologists to hope that the body's natural defense mechanisms might be employed to better resist established tumors. This optimism has extended to the long range plan that someday it may even be possible, at least theoretically, to immunize humans so that cancers never appear.
In the past 20 years, cancer biologists have become aware that, for many human and experimental tumors of animals, the cancer host's immune system does seem to have memory for the antigenic determinants that are present in a wide variety of natural and artificially induced tumors. Alternatively, interference is detected against the immune responses (e.g., suppression). The precise description of the means by which this immunologic memory comes into play in the hosts' susceptibility to antigenic tumor growth, however, has only recently begun to be elucidated.
For many years, cancer biologists have concerned themselves with detecting and characterizing new antigens which appear in or at the surface of tumor cells. Many such determinants occur on virally and chemically-induced tumors of animals. The types of antigens which appear in or on transformed cells as a result of abortive infection with oncogenic DNA-containing viruses, like SV40 virus, or infection with the RNA-containing retroviruses, can generally be shown to occur on different species of cells transformed by each of these viruses; however, each virus species induces unique antigens to those induced by different tumor viruses. Chemically induced neoplasms are often antigenic in their host, but may lack the viral associated transplantation antigens.
Best known among the tumor associated antigens which appear during the course of, or following, viral transformation of cells either in vitro or in vivo are (1) tumor associated cell surface antigens; (2) membrane associated tumor specific or tumor associated transplantation antigens; (3) intracellular antigens located primarily in the nucleus, termed tumor or T antigens, which may only function as an immunogen when the tumors become large and necrosis occurs; (4) virus associated antigens present on virus particles or on maturing virus in the cell membranes per se; and (5) embryonal or fetal antigens re-expressed at the cell surface and also in the extracellular fluid when SV40 transformed cells are cultured in vitro. The embryonal or fetal antigens can be detected with antibody and also by transplantation rejection tests. (This introduction is essentially taken from Coggin, Jr. and Ambrose, Methods in Cancer Research, Vol. 18, Chap. 10, pp. 371-389.)
There have been many attempts to develop antibodies, both polyclonal or monoclonal, against tumor antigens in the hope of preparing diagnostic and therapeutic reagents. Two types of immunizations have been carried out in the prior art with this hope in mind: xenogeneic and allogeneic immunizations. Xenogeneic immunization is the immunization of an animal, such as a mouse or rat, with tissue (e.g., tumor tissue) of an animal from a different species (e.g., human). Allogeneic immunization is obtained by immunizing an animal from a given species with tissue derived from an animal of the same species but of a different strain. Xenogeneic immunizations of mice which provide monoclonal antibodies against human neuroblastomas, for example, are described in Kennett, R. H., Chaapter 10 of Monoclonal Antibodies. Hybridomas: A New Dimension In Biological Analysis, pp. 115-168. Monoclonal antibodies raised xenogeneically against human leukemic cells derived from mouse splenocytes are described, for example, in Sato, et al., Development, Growth and Differentiation, 25:333-344 (1983).
There are several problems associated with antibodies (whether polyclonal or monoclonal) prepared by xenogeneic immunizations. Perhaps the most important one is the obvious difficulty of ever establishing absolute tumor specificity of such antibodies. Few monoclonal antibodies derived by xenogeneic immunization of mice or rats with a given human tumor tissue have demonstrated absolute specificity for that tumor class and many react with some normal adult human tissue (See, for example, Rosenberg, S. A., in Rosenberg Ed., Serological Analysis of Human Cancer Antigens, New York, Academic Press, 1980). These problems are derived from the fact that human tumor cells contain not only tumor specific antigens, but also a host of other normal, non-tumor related antigens when injected into histoincompatible hosts. Thus, polyclonal sera raised against tumor cells of a different species or strain is, by necessity, immunoreactive with both tumor specific and tumor nonspecific antigens. The possibility of obtaining monoclonal (Mc) antibodies against specific epitopes has raised the hope that some of the Mc's, randomly selected from hybridoma supernatants, would be truly tumor specific. This, however, is a tedious and laborious process, and provides poor guarantee of reproducibility of results. The results are "hit or miss" and are based on classic immunologic approaches for "raising" antibodies.
Over the last 10-15 years, there has developed, along a different line of cancer research, the possibility that the majority of malignant tumors of humans and rodents carry common embryonic determinants (EA) which can be immunogenic in the syngeneic host and are associated with the cell plasma membrane (See, for example, Coggin, Jr., Fetal Antigens and Cancer, Pittman, London (CIBA Foundation Symposium 96), pp. 28-54 (1983)). By the definition used herein, true EAs are uniquely expressed on germinal, embryonic and some fetal cell membranes, and are not expressed (immunogenic) in normal adult tissues nor in regenerating tissues. The immunological role of embryonic antigens in fetal development in utero is still obscure. It is known that maternal antibodies are produced in response to EA's expressed in utero. The biological product of the oncogenic process that leads to the re-expression of EA in the emerging malignant cell clone seems to be intimately associated with the promotion of tumor protected immune responses in the host. These responses mimic immune responses in pregnancy, which may serve to protect the EA.sup.+ fetus from maternal immune attack.
Embryonic antigens are indeed expressed by all classes of tumors of rodent and man. Some fetal antigen equivalent--onco-fetal antigens or OFA's--expressed in human tumors (for example, melanomas) appear to be segregated among distinct histologic classes, although common EA's have also been reported. It has been shown (Ambrose, K. R., et al., Nature 233:194 (1971); Coggin, J., et al., Advanced in Cancer Research 19:105 (1974) and Coggin, CIBA Symposium, supra) that rodent and human embryonic antigens shared as common immunogenic determinants and conserved in the evolution of species, are re-expressed as OFAs on human as well as rodent tumors. This idea originally arose from the observation that irradiated human fetal kidney cells as well as syngeneic irradiated hamster fetal cells could interrupt SV40 oncogenesis in hamsters. Adult human tissues were not similarly protective. This implies that rodent embryonic antigens are related to rodent oncofetal antigens, and that rodent embryonic antigens are identical with some human embryonic antigens. Since embryonic antigens present on human fetal kidney cells elicited protection against SV40-activated hamster embryonic antigens, at least some human embryonic antigens are equivalent to rodent oncofetal antigens.
Until recently, the nature of EAs was obscure since no reagents were available to immunochemically characterize them. A number of workers, however, have reported the detection of a variety of purported fetal antigens though none have been purified with monoclonal antibodies. Price, M. R., Soc. Trans., 2:650 (1974), detected a large uncharacterized fetal associated antigen with an estimated molecular weight of 100 kD in rat hepatomas. Evans et al., Can. Res., 39:2006 (1979), found that sarcomas of rats exhibited two onco-fetal antigens estimated to fall in the range of 3.5 and 10 kD. Dickinson et al., Br. J. Cancer, 29:425 (1974), reported that human tumor extracts of breast, cervix, vagina and omentum contained several small common proteins that appeared to be fetal associated, ranging in size from 16-18 kD. Jornvall et al., P.N.A.S. USA 79:287 (1982), reported the detection of a 53 kD onco-fetal protein which has been sequenced and found to share DNA binding properties with polyoma middle T antigen (55 kD). This transformation-associated protein was present in mid-gestation fetal cells in several species as well as in several human and adult tumor types evaluated.
While performing studies on the occurrence of rodent and/or human embryonic antigens on human tumor cells, some investigators have carried out syngeneic immunizations. A syngeneic immunization is one derived in an animal system devoid, by immunological methods, of detectable histo-incompatibility determinants. In other words, syngeneic immunizations are those obtained from immunizing a given animal with tissue from a genetically identical animal (i.e., some species, same strain).
Coggin, Jr., et al., The Journal of Immunology 105:524 (1970) and Coggin, et al., Adv. Can. Res., 19:105-165 (1974), showed that hamster and mouse fetal cells contained antigens cross-reactive with an SV40-induced sarcoma and on other viral and chemically-induced sarcomas, in that they stimulated an antibody. The antibody was synthesized when 10 day, but not 14 day, irradiated hamster fetal cells were injected into adult syngeneic hamsters, and these animals were subsequently found to exhibit immunity to SV40 tumor cell challenge. Non-irradiated fetal cells failed to induce transplantation immunity.
Hanna, Jr., Coggin, et al., Proceedings of the National Academy of Sciences, U.S.A. 68:1748 (1971), studied the suppressive effects of immunization with mouse fetal antigens on growth of cells infected with RLV virus and on plasma cell tumors. Young BALB/c male mice were primed at three week intervals with x-irradiated syngeneic embryo cells. The development of tumors was suppressed in the mice. Similar expressions were not observed in mice primed with neonatal or normal xenogeneic cells.
Ting, In Vitro 14:207 (1978), following the work described by Coggin, Jr. et al. (supra) employing sensitization to syngeneic fetus in hamsters and Balb/c mice, studied the expression of fetal antigens derived from mouse fetal cells. Antisera were produced by syngeneic immunization with 5,000 R x-irradiated tissues from mouse fetuses of one to two weeks gestation period. Fetal antigens were found to be retained even after five years in in vitro transformed cell lines from actual mouse fetal tissues.
All of these studies using syngeneic immunizations were carried out for the purpose of analyzing the presence of embryonic antigen on cells and their induction of immunity against tumors in the host. No production of hybridomas and Mc antibodies to fetal specific determinants on fetal tissues from a syngeneic host, has been described. Derivation of monoclonal antibodies reactive against any of the embryonic antigen determinants on the surface of hamster or mouse fetal cells of known gestational age has been expected to be problematic at best since such efforts would involve immunization within syngeneic systems. An additional complication would be that the usual course of multiple immunizations commonly reported in the monoclonal literature with soluble, crude embryonic antigen preparations have already been shown to activate T-suppressor lymphocytes, and to interfere with tumor transplantation immunity in a dose-dependent fashion (Weppner, W. A., et al., Cancer Research 40:1380 (1980)). The effects of hyperimmunization with syngeneic fetus on B lymphocyte activation were also unknown prior to this invention. Female rodents have been observed to develop cytostatic IgG to embryonic antigens cross- reactive with SV40 sarcoma cells when immunized directly with irradiated, syngeneic fetal cell preparations containing many disrupted fetal cells, but did not develop tumor resistance and presumably cytotoxic effector T cells under such conditions (Ambrose, K. R., supra; Coggin, J. H., Cancer Reserach, 39:2952 (1979).
Indeed, Shevinsky et al., Cell, 30:697-705 (1982), reported great difficulty in obtaining hybridomas with embryo-sensitized mouse or rat spleens. Only one embryo-specific monoclonal antibody (out of 2,000 clones in 14 fusions) could be obtained and reported. This, moreover, was obtained by xenogeneic immunization.
Thus, there were, prior to the present invention, several reasons in the art for expecting that syngeneic immunization might not work or even prove immunogenic to produce lymphocytes useful in the formation of hybridomas and of monoclonal antibodies applicable in diagnosis and/or therapy.
Because of the great need for obtaining immune reagents capable of high specificity with regard to detecting conserved EAs or OFAs in human tumors, the prior art prejudices were nevertheless ignored when the initial studies leading to the present invention were carried out.